DataSheet10_Notoginsenoside R1 Ameliorates Cardiac Lipotoxicity Through AMPK Signaling Pathway.ZIP

Aims: Cardiac lipotoxicity is the common consequence of lipid metabolism disorders in cardiomyocytes during development of heart failure (HF). Adenosine 5′monophosphate-activated protein kinase (AMPK) acts as an energy sensor and has a beneficial effect in reducing lipotoxicity. Notoginsenoside R1 (NGR1) is extracted from the traditional Chinese medicine Panax notoginseng (Burkill) F.H.Chen (P. notoginseng) and has definite cardioprotective effects. However, whether NGR1 can attenuate HF by mitigating lipotoxicity has not been elucidated yet. This study aimed to explore whether NGR1 plays a protective role against HF by ameliorating cardiac lipotoxicity via the AMPK pathway.
Methods: In this study, HF mice model was established by left anterior descending (LAD) ligation. palmitic acid (PA) stimulated H9C2 cell model was applied to clarify the effects and potential mechanism of NGR1 on lipotoxicity. In vivo, NGR1 (7.14 mg/kg/days) and positive drug (simvastatin: 2.9 mg/kg/days) were orally administered for 14 days. Echocardiography was applied to assess heart functions. Lipid levels were measured by Enzyme-linked immunosorbent assay (ELISA) and key proteins in the AMPK pathway were detected by western blots. In vitro, NGR1 (40 μmol/L) or Compound C (an inhibitor of AMPK, 10 μmol/L) was co-cultured with PA stimulation for 24 h in H9C2 cells. CCK-8 assay was used to detect cell viability. Key lipotoxicity-related proteins were detected by western blots and the LipidTOX™ neutral lipid stains were used to assess lipid accumulation. In addition, Apoptosis was assessed by Hoechst/PI staining.
Results: NGR1 could significantly improve the cardiac function and myocardial injury in mice with HF and up-regulate the expression of p-AMPK. Impressively, NGR1 inhibited the synthesis of diacylglycerol (DAG) and ceramide and promoted fatty acid oxidation (FAO) in vivo. Moreover, NGR1 significantly promoted expression of CPT-1A, the key enzyme in FAO pathway, and down-regulated the expression of GPAT and SPT, which were the key enzymes catalyzing production of DAG and ceramide. In vitro experiments showed that NGR1 could significantly attenuate lipid accumulation in PA-induced H9C2 cells and the Hoechst/PI staining results showed that NGR1 ameliorated lipotoxicity-induced apoptosis in PA-stimulated H9C2 cell model. Furthermore, co-treatment with inhibitor of AMPK abrogated the protective effects of NGR1. The regulative effects of NGR1 on lipid metabolism were also reversed by AMPK inhibitor.
Conclusion: NGR1 could significantly improve the heart function of mice with HF and reduce cardiac lipotoxicity. The cardio-protective effects of NGR1 are mediated by the activation of AMPK pathway.

研究目的：心力衰竭（heart failure, HF）发生过程中，心肌细胞脂质代谢紊乱常引发心肌脂毒性。腺苷5′-单磷酸活化蛋白激酶（adenosine 5′monophosphate-activated protein kinase, AMPK）作为能量感受器，在减轻脂毒性方面具有有益作用。三七皂苷R1（notoginsenoside R1, NGR1）提取自中药三七[Panax notoginseng (Burkill) F.H.Chen, P. notoginseng]，已被证实具有明确的心脏保护作用。但NGR1是否可通过减轻心肌脂毒性改善心力衰竭，目前尚未阐明。本研究旨在探讨NGR1是否可通过AMPK通路改善心肌脂毒性，从而发挥抗心力衰竭的保护作用。
研究方法：本研究通过结扎左前降支（left anterior descending, LAD）构建心力衰竭小鼠模型，采用棕榈酸（palmitic acid, PA）诱导H9C2细胞构建脂毒性细胞模型，以明确NGR1对脂毒性的作用及潜在机制。体内实验中，小鼠每日经口给予NGR1（7.14 mg/kg）及阳性药物辛伐他汀（simvastatin, 2.9 mg/kg），连续给药14天。通过超声心动图评估心脏功能，采用酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）检测脂质水平，蛋白质免疫印迹（western blots）检测AMPK通路关键蛋白的表达。体外实验中，将NGR1（40 μmol/L）或AMPK抑制剂Compound C（10 μmol/L）与PA共同孵育H9C2细胞24小时。采用CCK-8法检测细胞活力，蛋白质免疫印迹检测脂毒性相关关键蛋白，使用LipidTOX™中性脂质染色剂评估脂质蓄积情况，此外通过Hoechst/PI染色检测细胞凋亡。
研究结果：NGR1可显著改善心力衰竭小鼠的心脏功能与心肌损伤，并上调p-AMPK的表达。值得注意的是，NGR1在体内可抑制二酰基甘油（diacylglycerol, DAG）与神经酰胺的合成，同时促进脂肪酸氧化（fatty acid oxidation, FAO）。此外，NGR1可显著上调脂肪酸氧化通路关键酶CPT-1A的表达，下调催化二酰基甘油与神经酰胺合成的关键酶GPAT与SPT的表达。体外实验结果显示，NGR1可显著减轻棕榈酸诱导的H9C2细胞脂质蓄积；Hoechst/PI染色结果表明，NGR1可改善棕榈酸刺激下H9C2细胞的脂毒性诱导凋亡。进一步实验显示，联合使用AMPK抑制剂可抵消NGR1的保护作用，且NGR1对脂质代谢的调控作用也可被AMPK抑制剂逆转。
研究结论：本研究表明，NGR1可显著改善心力衰竭小鼠的心脏功能，减轻心肌脂毒性，其心脏保护作用通过激活AMPK通路介导。